Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81.

Petros Batsios,Denis Larochelle,Xiang Ren,Ralph Gräf,Otto Baumann

doi:10.3390/cells5010013

Abstract

The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.

Highlights

In animal cells, the nuclear envelope is composed of an outer nuclear membrane (ONM), which is continuous with the endoplasmic reticulum, and an inner nuclear membrane (INM) supported by the nuclear lamina [1]
Among the INM proteins, we have recently shown by proximity-dependent biotin identification (BioID) that NE81 displays the conserved interaction of Sun1 with lamins [19]
In this paper we provide the first characterization of a MAN1-like HeH-family protein, Src1, in an amoebozoan, and show by light and electron microscopy that Src1 is an INM protein that interacts with the Dictyostelium lamin NE81 in BioID and mis-localization assays

Summary

Introduction

The nuclear envelope is composed of an outer nuclear membrane (ONM), which is continuous with the endoplasmic reticulum, and an inner nuclear membrane (INM) supported by the nuclear lamina [1]. The inner and outer nuclear membrane are separated by the perinuclear space and contiguous with each other at nuclear pore complexes, which are in contact with the nuclear lamina. The latter refers to a protein network consisting of inner nuclear membrane proteins and a network of type V intermediate filaments called lamins. In A-type lamins the prenyl group together with the last 15 amino acids is cleaved off prior to filament assembly, while it persists in B-type lamins

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